1. Field of the Invention
This invention relates to a polyhydroxyalkanoate (hereinafter, referred to as a xe2x80x9cPHAxe2x80x9d) synthase, a gene encoding the PHA synthase, a recombinant vector containing the gene, a transformant capable of expressing the PHA synthase which has been transformed by the recombinant vector, a process for producing the PHA synthase utilizing the transformant, and a process for preparing the PHA utilizing the transformant. In particular, this invention relates to a microorganism-derived PHA synthase capable of producing a polyhydroxyalkanoate and a gene encoding the PHA synthase utilized for expressing the PHA synthase by transformation.
2. Related Background Art
There have been reported a number of microorganisms producing poly-3-hydroxybutyric acid (PHB) or another PHA and storing it therein (xe2x80x9cBiodegradable Plastic Handbookxe2x80x9d, edited by Biodegradative Plastic Research Society, NTS Co. Ltd., p.178-197). These polymers may be, as conventional plastics, used for producing a variety of products by, for example, melt-processing. Since they are biodegradable, they have an advantage that they can be completely degraded by microorganisms in the natural environment, and they do not cause pollution due to remaining in the natural environment like many conventional polymer compounds. Furthermore, they are excellently biocompatible, and thus are expected to be used in applications such as a medical soft member.
It is known that a composition and a structure of such a PHA produced by a microorganism may considerably vary depending on the type of a microorganism used for the production, a culture-medium composition and culturing conditions. Investigations have been, therefore, mainly focused on controlling such a composition or structure for the purpose of improving physical properties of a PHA.
For example, Japanese Patent Application Nos. 7-14352 and 8-19227 and Japanese Examined Publication No. 6-15604 described that Alcaligenes eutropus H16 (ATCC No. 17699) and its variants may produce 3-hydroxybutyric acid (3HB) and its copolymer with 3-hydroxyvaleric acid (3HV) with various composition ratios by changing a carbon source during culturing.
Japanese Patent Publication No. 26-42937 discloses that PHA in which a monomer unit is 3-hydroxyalkanoate with 6 to 12 carbon atoms may be produced by supplying a non-cyclic aliphatic hydrocarbon as a carbon source to Pseudomonas oleovorans (ATCC No. 293-47).
Japanese Patent Application Laid-Open No. 5-7492 discloses methods in which Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp. are contacted with a primary alcohol with 3 to 7 carbon atoms to produce a copolymer of 3HB and 3HV.
Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065 disclose that Aeromonas caviae is cultured using oleic acid or olive oil as a carbon source to produce a two-component copolymer of 3HB and 3-hydroxyhexanoic acid (3HHx).
Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IF013852 is cultured using gluconic acid and 1,4-butanediol as carbon sources to produce a polyester having 3HB and 4-hydroxybutyric acid as monomer units.
Furthermore, it is reported that certain microorganisms produce PHAs having a variety of substituents such as unsaturated hydrocarbon, ester, aryl (aromatic), and cyans groups, halogenated hydrocarbon and epoxide. Recently, there have been attempts for improving physical properties of a PHA produced by a microorganism using such a procedure. For example, Makromol. Chem., 191, 1957-1965 (1990); Macromolecules, 24, 5256-5260 (1991); and Chirality, 3, 492-494 (1991) describe production of a PHA comprising 3-hydroxy-5-phenylvaleric acid (3HPV) as a monomer unit by Pseudomonas oleovorans, where variations in polymer physical properties probably due to the presence of 3HPV were observed.
As described above, microorganism-produced PHAs with various combinations of composition and structure have been obtained by varying factors such as the type of a microorganism used, a culture medium composition and culturing conditions. Each microorganism has an intrinsic PHA synthase with a substrate specificity which is significantly different from others. Thus, it has been difficult to produce PHAs comprising different monomer units suitable to a variety of applications using known microorganisms or PHA synthases in such known microorganisms.
Meanwhile, as described above, a PHA having a variety of substituents in its side chains may be expected to be a xe2x80x9cfunctional polymerxe2x80x9d having significantly useful functions and properties owing to the properties of the introduced substituents. It is, therefore, extremely useful and important to search and develop a microorganism which can produce and store a very useful polymer having both such functionality and biodegradability. Furthermore, identification of a PHA synthase involved in production of the highly useful PHA and obtaining a gene encoding the PHA synthase may allow us to produce a novel transformed microorganism capable of producing a desired PHA. That is, constructing a recombinant vector comprising a gene encoding a PHA synthase and providing a microorganism transformed by the recombinant vector may allow us to prepare a PHA using the transformed microorganism or to express a recombinant type of PHA synthase. As described above, it may be important that a transformed microorganism is used to prepare a desired PHA for providing a highly useful tool for improving a productivity for the PHA and for promoting utilization of the PHA.
Objects of this invention which can solve the above problems are to search a novel microorganism capable of producing and storing in microorganisms a PHA having a novel side-chain structure, to identify an enzyme protein related to the ability of producing the novel PHA, i.e., a novel PHA synthase, and to determine a gene encoding its amino acid sequence. More specifically, an object of the present invention is to provide a novel PHA synthase derived from a microorganism producing a PHA having a novel side chain structure and a DNA encoding its amino acid sequence. Another object of this invention is to provide a recombinant vector to which a DNA encoding an available PHA synthase is introduced and which is used for transformation of a microorganism and a transformed microorganism produced using the recombinant vector. A further object of this invention is to provide a process for expressing and producing a recombinant PHA synthase in the transformed microorganism and a process for preparing a desired PHA using the transformed microorganism.
Still another object of this invention is to provide a modified PHA synthase in which its amino acid sequence is modified as long as an enzyme activity is not affected in expression of the recombinant PHA synthase in the transformed microorganism as described above and a DNA encoding the modified amino acid sequence.
For developing a PHA having a novel side-chain structure useful as, for example, a device material or a medical material aiming at solving the above problems, the inventors have searched a novel microorganism capable of producing and storing the desired PHA therein. Additionally, the inventors have intensely investigated selected novel microorganisms producing a novel PHA for identifying a PHA synthase involved in production of the novel PHA and for obtaining a gene encoding the PHA synthase. Furthermore, the inventors have conducted investigation for constructing a recombinant vector with a gene for the obtained PHA synthase, transforming a host microorganism using the recombinant vector, expressing a recombinant PHA synthase in the transformed microorganism obtained and determining production of the desired PHA.
In the course of the above investigation, the inventors synthesized 5-(4-fluorophenyl) valeric acid (FPVA) represented by formula (II): 
and separated from a soil a novel microorganism capable of converting the above compound (II) as a starting material (substrate) into corresponding 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) represented by formula (III): 
and producing and storing a novel PHA with a monomer unit represented by formula (I): 
derived from 3HFPV. The novel microorganism separated is designated as P161 strain. The inventors have also found that in addition to the above enzymatic activity for converting FPVA into 3HFPV, the P161 strain may also use 4-phenoxybutyric acid (PXBA) represented by formula (IV): 
as a starting material (substrate) to convert it into 3-hydroxy-4-phenoxybutyric acid (3HPxB) represented by formula (V): 
xe2x80x83and to produce and store a PHA with a monomer unit represented by formula (VI): 
derived from 3HPxB. There have been no reports for microbial production of a PHA comprising 3HPxB as a monomer unit using PXBA as a substrate or for microbial production of a PHA comprising 3HPxB as a sole phenoxy-containing monomer unit.
An example of a microorganism capable of producing and storing a PHA with a monomer unit represented by formula (VI) derived from 3HPxB using a substrate other than PxBA is Pseudomonas oleovorans using 8-phenoxyoctanoic acid (PxOA) as a substrate described in Macromolecules, 29, 3432-3435, 1996. In Pseudomonas oleovorans, 8-phenoxyoctanoic acid (PxOA) is used as a substrate, which is totally different from the enzymatic reaction in P161 strain where PxBA is used as a substrate to produce a PHA with a monomer unit represented by formula (VI) derived from a corresponding 3HPxB. In addition, for the composition of a PHA produced, the reported process using Pseudomonas oleovorans provides a copolymer consisting of three monomer units, i.e., 3-hydroxy-8-phenoxyoctanoic acid corresponding to PxOA as a substrate, 3-hydroxy-6-phenoxyhexanoic acid as a byproduct derived from a metabolite of the substrate, and the desired 3HPxB. On the other hand, a process where P161 strain acts on the substrate PxBA provides a PHA with 3HPxB derived from PXBA as a sole phenoxy-containing monomer unit. Taking the compositions of the PHAs also into consideration, it seems that there is fundamental difference in substrate specificity of a PHA synthase between Pseudomonas oleovorans used in the above process reported and P161 strain. That is, a PHA synthase produced by P161 strain is more preferable for production of a PHA with 3HPxB as a monomer unit.
Furthermore, the inventors have found that P161 strain can use 6-phenylhexanoic acid (PHxA) represented by formula (VII): 
as a starting material (substrate) to convert it into corresponding 3-hydroxy-6-phenylhexanoic acid (3HPHx) represented by formula (VIII): 
and to produce and store a novel PHA with a monomer unit represented by formula (IX): 
derived from 3HPHx.
Microbiological properties of P161 strain are as follows.
 less than Microbiological Properties of P161 Strain greater than 
Morphologic Properties
Cell shape and size: Sphere, xcfx860.6 xcexcm
Bacilliform, 0.6 xcexcmxc3x971.5 to 2.0 xcexcm
Cell polymorphism: Yes (elongation)
Motility: Yes
Sporulation: No
Gram stainability: Negative
Colonization: Circular, smooth in the overall periphery, low convex, smooth surface, pale yellow
Physiological Properties
Catalase: Positive
Oxidase: Positive
O/F test: oxidized form
Reduction of a nitrate: Positive
Indole formation: Negative
Acidification of dextrose: Negative
Arginine dihydrolase: Positive
Urease: Negative
Esculin hydrolysis: Negative
Gelatin hydrolysis: Negative
xcex2-Galactosidase: Negative
Fluorochrome production on King""s B agar: Positive
Substrate Assimilation Ability
Dextrose: Positive
L-Arabinose: Positive
D-Mannose: Positive
D-Mannitol: Positive
N-Acetyl-D-glucosamine: Positive
Maltose: Negative
Potassium gluconate: Positive
n-Capric acid: Positive
Adipic acid: Negative
dl-Malic acid: Positive
Sodium citrate: Positive
Phenyl acetate: Positive
From these microbiological properties, the inventors have attempted to categorize P161 strain according to Bergey""s Manual of Systematic Bacteriology, Volume 1 (1984) and Bergey""s Manual of Determinative Bacteriology 9th ed. (1994) to determine that the strain belongs to Pseudomonas sp. Its taxonomic position could not been determined from these microbiological properties.
Thus, for categorizing P161 strain from its genetic properties, the inventors sequenced its 16S rRNA (SEQ ID NO. 5) and compared its homology with the sequence of a 16S rRNA in a known Pseudomonas sp. microorganism. The results indicate quite higher homology in a 16S rRNA sequence between P161 strain and a known Pseudomonas jessenii. Furthermore, microbiological properties described for the known Peudomonas jessenii in System. Appl. Microbiol., 20, 137-149 (1997) and System. Appl. Microbiol., 22, 45-58 (1999) was compared with those for P161 strain and observed considerable homology. From these results, it was judged to be proper to categorize P161 strain in Pseudomonas jessenii, and thus it is designated as Pseudomonas jessenii P161. There have been no reports on a strain in Pseudomonas jessenii capable of producing a PHA as exhibited by P161 strain. The inventors have, therefore, determined that P161 strain is a novel microorganism. The applicant deposited Pseudomonas jessenii P161 to Patent Microorganism Depository Center in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under the deposition number of FERM P-17445. P161 strain has been internationally deposited on the basis of the Budapest Treaty, and its international accession number is xe2x80x9cFERM BP-7376xe2x80x9d.
The inventors achieved cloning a gene for a PHA synthase from the novel microorganism P161 strain and sequenced the gene. The inventors also determined an amino acid sequence for the PHA synthase encoded by the gene. Based on the above observation, the present invention was achieved.
Specifically, a PHA synthase of the present invention is a polyhydroxyalkanoate synthase having an amino acid sequence of SEQ ID NO. 1 or 3. Furthermore, the PHA synthase of the present invention may be a PHA synthase substantially retaining the amino acid sequence of SEQ ID NO. 1 and having a modified amino acid sequence where amino acids are deleted, substituted or added as long as it does not deteriorate an activity as the polyhydroxyalkanoate synthase, or a PHA synthase substantially retaining the amino acid sequence of SEQ ID NO. 3 and having a modified amino acid sequence where amino acids are deleted, substituted or added as long as it does not deteriorate activity as the polyhydroxyalkanoate synthase.
A PHA synthase gene of the present invention is a gene for a polyhydroxyalkanoate synthase comprising a DNA encoding the amino acid sequence of SEQ ID NO. 1 or the sequence of its modified amino acid, or a gene for a polyhydroxyalkanoate synthase comprising a DNA encoding the amino acid sequence of SEQ ID NO. 3 or the sequence of its modified amino acid. Embodiments of a PHA synthase gene of the present invention derived from a genome gene in P161 strain include a PHA synthase gene comprising a DNA sequence of SEQ ID NO. 2 as a DNA encoding the amino acid sequence of SEQ ID NO. 1 and a PHA synthase gene comprising a DNA sequence of SEQ ID NO. 4 as a DNA encoding the amino acid sequence of SEQ ID NO. 3.
This invention also provides a recombinant vector comprising a gene DNA encoding the above amino acid sequence as a polyhydroxyalkanoate synthase gene. This invention also provides a transformed microorganism transformed by introducing a recombinant vector adapted to a host.
The present invention also provides a process for preparing a polyhydroxyalkanoate comprising the steps of culturing the transformed microorganism to which a recombinant vector has been introduced in a culture medium containing a substrate for a polyhydroxyalkanoate synthase and collecting the polyhydroxyalkanoate from the culture preparation. The present invention also provides a process for producing a polyhydroxyalkanoate comprising the steps of culturing the transformed microorganism to which a recombinant vector has been introduced and making the transformed microorganism produce the polyhydroxyalkanoate.
A preferable process for producing a polyhydroxyalkanoate may utilize substrate specificity characteristic of a polyhydroxyalkanoate synthase derived from P161 strain: for example, preparation of a polyhydroxyalkanoate comprising a monomer unit represented by formula (I) derived from 3HFPV utilizing the above transformed microorganism; preparation of a polyhydroxyalkanoate comprising a monomer unit represented by formula (VI) derived from 3HPxB, or preparation of a polyhydroxyalkanoate comprising a monomer unit represented by formula (IX) derived from 3HPHx.
A PHA synthase and a gene encoding the PHA synthase of the present invention are derived from a novel microorganism, Pseudomonas jessenii P161 strain and exhibits such substrate specificity that it selectively produces a PHA comprising a monomer unit having a novel side chain structure. A recombinant vector comprising the PHA synthase gene and a microorganism transformed by the recombinant vector are capable of producing a PHA exhibiting substrate specificity similar to Pseudomonas jessenii P161. Thus, a PHA synthase gene of this invention encodes an enzyme which permits preparation of a PHA selectively comprising a monomer unit having a novel side-chain structure and allows us to create a transformed microorganism useful for preparing a PHA having various useful physical properties which may be expected to be applied to a functional polymer.